Structural transition of GP64 triggered by a pH-sensitive multi-histidine switch

Abstract

The fusion of viruses with cellular membranes is a critical step in the life cycle of enveloped viruses. This process is facilitated by viral fusion proteins, many of which are conformationally pH-sensitive. The specifics of how changes in pH initiate this fusion have remained largely elusive. This study presents the cryo-electron microscopy (cryo-EM) structures of a prototype class III fusion protein, GP64, in its prefusion and early intermediate states, revealing the structural intermediates accompanying the membrane fusion process. The structures identify the involvement of a pH-sensitive switch, comprising H23, H245, and H304, in sensing the low pH that triggers the initial step of membrane fusion. The pH sensing role of this switch is corroborated by assays of cell-cell syncytium formation and dual dye-labeling. The findings demonstrate that coordination between multiple histidine residues acts as a pH sensor and activator. The involvement of a multi-histidine switch in viral fusion is applicable to fusogens of human-infecting thogotoviruses and other viruses, which could lead to strategies for developing anti-viral therapies and vaccines.

Fig. 1. Overall structures of GP64 in prefusion and early intermediate states.

a Linear representation of GP64 (the color code in (a) is also used for domains and subdomains in (b)). The signal peptide (residues 1−20), part of domain III (residues 21 and 22), and the C-terminal region (residues 488−512) are not observed in the structure and are colored white. The transmembrane region (TM) is labeled using a checkerboard pattern. b Density map (left) and ribbon diagram (right) of a prefusion GP64 monomer. c Ribbon diagram of a GP64 trimer in the prefusion state. Protomers A, B, and C are colored red, cyan, and dark blue, respectively. d Ribbon diagram of GP64 in the intermediate state; the coloring scheme is the same as in (c). Protomer A adopts an early intermediate state, while protomers B and C are almost in the prefusion conformation.

Fig. 2. Structural details of prefusion GP64.

Domains and subdomains in (a–d) are color-coded as in Fig. 1c. Superscripts A, B, and C on labels indicate protomers A, B, and C, respectively. Salt bridges, hydrogen bonds, and van der Waals contacts are indicated by dotted magenta, cyan, and dark orange lines respectively. a Side and top views showing intra- and interprotomer interactions between the basic loop (BL) of protomer A and the BLs/B helices of adjacent protomers. BL forms a stable structure through these interactions. b Interactions between domains II and III in prefusion (left) and postfusion (right) (PDB code: 3DUZ) states. c Ribbon representation of the helix D trimer in the prefusion state viewed parallel to the membrane (left) and perpendicular to the membrane (right). d Ribbon representation of PTMD and TMD trimers of prefusion GP64. e Ribbon (left) and surface (right) representations of domain I, PTMD, and TMD showing interactions between fusion loop-1 and PTMD. Fusion loops are ~10 Å above the viral envelope membrane. Residues are colored as in Fig. 1a (left), or based on hydrophobicity (right).

Fig. 3. Structural rearrangements of GP64 during membrane fusion.

a–c Side, bottom, and top views of GP64 in prefusion (a), intermediate-M (b), and postfusion (c) states showing protein conformational changes. The coloring scheme is the same as that in Fig. 1a. For clarity, the C-terminal region (residues 437–487) is omitted in the bottom and top views of GP64. d Comparison of CTS1 and CTS2 in prefusion (left) and postfusion (right) states. The coloring scheme is the same as that in Fig. 1c. Superscripts A, B, and C on labels indicate protomers A, B, and C, respectively.

Fig. 4. Construction and expression of His-to-Ala substituted GP64s.

a pH-sensitive interfaces of GP64. Three clusters of histidine residues in the prefusion state are shown in regions indicated by black rectangles (left). The same residues are shown in the postfusion state (right). Salt bridges and hydrogen bonds are indicated by dotted magenta and cyan lines. b Schematic diagram of GP64 constructs. T, M, and L represent top, middle, and lower regions, respectively, within the trimer interface of prefusion GP64. c Western blotting analysis of transient expression and trimerization of WT and modified GP64s. β-tubulin served as a loading control. d Relative cell surface levels of GP64s were measured via cELISA. A standard curve was generated by transfecting cells with decreasing quantities of a plasmid expressing WT GP64, which is shown on the left side of the panel. Error bars represent standard deviation (SD) from the mean of independent replicates (n = 3).

Fig. 5. Membrane fusion and low pH-induced conformational changes in GP64s.

a Cell-cell syncytium formation assay. At 36 h p.t., transfected cells were exposed to low pH to induce syncytium formation and fusion activity was measured. Arrows indicate the syncytial mass (≥5 nuclei). Error bars represent the SD from the mean of independent replicates (n = 3). b Hemifusion and pore formation mediated by fusion-deficient GP64s. The top panel shows hemifusion and pore formation mediated by the modified GP64s. The bottom panel shows dye transfer efficiency. The dye transfer efficiency was estimated as the ratio of the number of R18-transferred or calcein-AM-transferred Sf9 cells to the number of Sf9 cells with bound RBCs. Error bars represent the SD of means of independent replicates (n = 3). c Analysis of low pH-triggered conformational changes in fusion-deficient GP64 constructs. At 36 h p.t., the cell-surface-localized GP64 proteins subjected to low pH triggering were measured by cELISA using MAb AcV1 and then normalized against WT at pH 7.0. Error bars represent the SD from the mean of independent replicates (n = 3). Scale bar, 50 μm.

Fig. 6. Schematic cartoon diagrams of GP64 rearrangements.

a In the prefusion state (at neutral pH), domain I aligns with the β-sheets of domain IV and PTMD, while domain II aligns with the initial part of helix B of domain III. The fusion loops located at the tips of domain I are initially anchored onto PTMD. CTS1 of each protomer fits into the groove of the central helices. CTS2 forms a curled structure positioned between domains I/II and the central domains (III and IV). b During the transformation, rotation of domain I causes the disengagement of domains I/II from domains III/IV and the fusion loops from PTMD. This repositioning renders CTS2 accessible (This might indicate an early intermediate state). c Subsequently, domains I and II detach from the central core and rotate by 180°, and domain II aligns with the central helix again but in a reverse manner. The fusion loops attach to the host membrane. d CTS1 moves away from the grooves of the central helices, allowing CTS2 to straighten and snugly fit into this groove, driving foldback of the extended intermediate. e, f This action prompts helix D to ascend alongside domains I and II, positioning both PTMD and TMD, to which it is connected, at the same end of the molecule, causing the fusion of virus and host membranes. c–e depict later intermediate states, while (f) represents the postfusion state. The yellow dots represent the approximate locations of the key histidines H23, H245, and H304, respectively; while the black dashed lines indicate the interactions between these histidines and adjacent residues.